1. Introduction
The purification of immunoglobulin from mammalian sera for therapeutic and research applications is an issue of considerable industrial, medical, and economical value. Polyclonal antibody-based therapeutics exhibit polyvalent interactions against multiple epitopes and targets and are therefore best suited for the prevention or treatment of some diseases. Plasma-derived polyclonal intravenous immunoglobulin (IVIG) preparations have been successfully applied to the prophylactic prevention of infectious diseases in immunodeficient patients and find increasing use against autoimmune and inflammatory problems. To date, IVIG is the major plasma product on the global blood product market, with a steadily increasing annual consumption. Polyclonal antibodies derived from animal plasma are also currently employed in research for producing immunoassays and to design therapeutic and diagnostic tools.
Affinity purification of polyclonal antibodies from mammalian sera is currently mostly based upon the use of protein ligands, such as Protein A and Protein G. These protein ligands, however, suffer from several drawbacks, such as 1) high cost ($15,000-20,000 per liter of adsorbent), 2) low chemical and biochemical stability, 3) immunogenicity, with the consequent risks associated to the leaching of ligand fragments in the product mainstream, and 4) harsh elution conditions, due to the high binding affinity, which threatens the bioactivity of the eluted protein. Further, Protein A does not bind human IgG3 and several animal immunoglobulins. Protein G, while binding all human IgG subclasses, shows also considerable binding of albumin, which is a major protein in human and animal plasma and its fractions.
To overcome these issues, synthetic ligands based on peptides, amino acids, triazine scaffolds and thiophillic compounds have been suggested for purification of antibodies. Our research group has identified three hexapeptide ligands, HWRGWV, HYFKFD and HFRRHL (SEQ ID NOS:1-3), which bind IgG through the Fc portion, thus mimicking the binding mechanism of Protein A. In particular, the peptide HWRGWV (SEQ ID NO: 1) was further characterized for its ability to isolate IgG from a variety of complex sources, including cell culture media, CHO cell culture supernatants, transgenic milk and whey, plant extract, and Cohn fraction II+III of human plasma. The product yields and purities that resulted from these experiments were always comparable to those obtained with Protein A media.
However, an issue that both protein ligands and synthetic peptide ligands face when used for the purification of polyclonal antibodies from human plasma is the action of proteolytic enzymes present therein, in particular trypsin and α-chymotrypsin. Trypsin is a serin protease that cleaves peptide chains at the carboxyl side of lysine and arginine residues. Chymotrypsin cleaves peptide chains at the carboxyl side of hydrophobic residues, such as tyrosine, tryptophan, and phenylalanine. Upon prolonged exposure and/or repeated to mammalian sera, either protein or peptide ligands are degraded by these endoproteases. To prevent degradation of Protein A by these endoproteases and hence the decrease of the lifetime of costly affinity resin, enzyme inhibitors are added to feed before injection. These inhibitors, however, represent a considerable additional cost themselves.